ORIGINAL ARTICLE Acidic lipase Lip I.3 from a Pseudomonas fluorescens-like strain displays unusual properties and shows activity on secondary alcohols P. Panizza 1,2 , N. Syfantou 1 , F.I.J. Pastor 1 , S. Rodr  ıguez 2 and P. D  ıaz 1 1 Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain 2 Bioscience Department, Faculty of Chemistry, Universidad de la Rep ublica, Montevideo, Uruguay Keywords biocatalysis, lipase, Pseudomonas, secondary alcohols, subfamily I.3. Correspondence Paola Panizza, Department of Microbiology, Faculty of Biology, University of Barcelona, Av. Diagonal 645, 08028-Barcelona, Spain. E-mail: ppanizza@fq.edu.uy 2013/1792: received 5 October 2012, revised 16 November 2012 and accepted 23 November 201220122012 doi:10.1111/jam.12089 Abstract Aims: Identification, cloning, expression and characterization of a novel lipase – Lip I.3 – from strain Pseudomonas CR-611. Methods and Results: The corresponding gene was identified and isolated by PCR-amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0-1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C-terminal type I secretion signal and several putative Ca 2+ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca 2+ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols. Conclusions: A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts. Significance and Impact of the Study: The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis. Introduction Lipases (EC 3.1.1.3) are enzymes that hydrolyze the car- boxyl ester bonds in acyl-glycerides. Microbial lipases are the second largest group of industrial biocatalysts after bacterial amylolytic enzymes (Guncheva and Zhiryakova 2011). They have found numerous applications within biotechnology industries such as food technology, deter- gent formulation, chemical industry and biomedical sci- ences (Hasan et al. 2006; Guncheva and Zhiryakova 2011). More interestingly, some lipases show high regio and stereoselectivity, rendering them important tools in the synthesis of chiral compounds for the pharmaceutical industry (Patel 2006; Ghanem 2007; Turner 2010). The increasing demand for environmentally benign industrial processes, as well as the rising need for stereoselective synthetic routes, has prompted the search for novel enzymes to broaden the scope of biocatalytic tools and applications (Steele et al. 2009; Zhang et al. 2009). Most lipases used in biocatalysis are microbial enzymes isolated from both, fungi and bacteria. Amongst the fungal lipases, those isolated from Candida antarctica, Candida rugosa, Geotrichum and Rhizopus have found extensive applications in organic synthesis (Lambusta et al. 2003; Dom  ınguez de Mar  ıa et al. 2005, 2006; Ghanem 2007). Amongst the bacterial lipases, the most 722 Journal of Applied Microbiology 114, 722--732 © 2013 The Society for Applied Microbiology Journal of Applied Microbiology ISSN 1364-5072